Nanopatterned Extracellular Matrices Enable Cell-Based Assays with a Mass Spectrometric Readout
[Image: see text] Cell-based assays are finding wider use in evaluating compounds in primary screens for drug development, yet it is still challenging to measure enzymatic activities as an end point in a cell-based assay. This paper reports a strategy that combines state-of-the-art cantilever free p...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2017
|
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501326/ https://www.ncbi.nlm.nih.gov/pubmed/28120616 http://dx.doi.org/10.1021/acs.nanolett.6b04176 |
_version_ | 1783248767454019584 |
---|---|
author | Cabezas, Maria D. Mirkin, Chad A. Mrksich, Milan |
author_facet | Cabezas, Maria D. Mirkin, Chad A. Mrksich, Milan |
author_sort | Cabezas, Maria D. |
collection | PubMed |
description | [Image: see text] Cell-based assays are finding wider use in evaluating compounds in primary screens for drug development, yet it is still challenging to measure enzymatic activities as an end point in a cell-based assay. This paper reports a strategy that combines state-of-the-art cantilever free polymer pen lithography (PPL) with self-assembled monolayer laser desorption–ionization (SAMDI) mass spectrometry to guide cell localization and measure cellular enzymatic activities. Experiments are conducted with a 384 spot array, in which each spot is composed of ∼400 nanoarrays and each array has a 10 × 10 arrangement of 750 nm features that present extracellular matrix (ECM) proteins surrounded by an immobilized phosphopeptide. Cells attach to the individual nanoarrays, where they can be cultured and treated with small molecules, after which the media is removed and the cells are lysed. Phosphatase enzymes in the proximal lysate can then act on the immobilized phosphopeptide substrate to convert it to the dephosphorylated form. After the lysate is removed, the array is analyzed by SAMDI mass spectrometry to identify the extent of dephosphorylation and, therefore, the amount of enzyme activity in the cell. This novel approach of using nanopatterning to mediate cell adhesion and SAMDI to record enzyme activities in the proximal lysate will enable a broad range of cellular assays for applications in drug discovery and research not possible with conventional strategies. |
format | Online Article Text |
id | pubmed-5501326 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-55013262017-07-07 Nanopatterned Extracellular Matrices Enable Cell-Based Assays with a Mass Spectrometric Readout Cabezas, Maria D. Mirkin, Chad A. Mrksich, Milan Nano Lett [Image: see text] Cell-based assays are finding wider use in evaluating compounds in primary screens for drug development, yet it is still challenging to measure enzymatic activities as an end point in a cell-based assay. This paper reports a strategy that combines state-of-the-art cantilever free polymer pen lithography (PPL) with self-assembled monolayer laser desorption–ionization (SAMDI) mass spectrometry to guide cell localization and measure cellular enzymatic activities. Experiments are conducted with a 384 spot array, in which each spot is composed of ∼400 nanoarrays and each array has a 10 × 10 arrangement of 750 nm features that present extracellular matrix (ECM) proteins surrounded by an immobilized phosphopeptide. Cells attach to the individual nanoarrays, where they can be cultured and treated with small molecules, after which the media is removed and the cells are lysed. Phosphatase enzymes in the proximal lysate can then act on the immobilized phosphopeptide substrate to convert it to the dephosphorylated form. After the lysate is removed, the array is analyzed by SAMDI mass spectrometry to identify the extent of dephosphorylation and, therefore, the amount of enzyme activity in the cell. This novel approach of using nanopatterning to mediate cell adhesion and SAMDI to record enzyme activities in the proximal lysate will enable a broad range of cellular assays for applications in drug discovery and research not possible with conventional strategies. American Chemical Society 2017-01-25 2017-03-08 /pmc/articles/PMC5501326/ /pubmed/28120616 http://dx.doi.org/10.1021/acs.nanolett.6b04176 Text en Copyright © 2017 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
spellingShingle | Cabezas, Maria D. Mirkin, Chad A. Mrksich, Milan Nanopatterned Extracellular Matrices Enable Cell-Based Assays with a Mass Spectrometric Readout |
title | Nanopatterned Extracellular Matrices Enable Cell-Based
Assays with a Mass Spectrometric Readout |
title_full | Nanopatterned Extracellular Matrices Enable Cell-Based
Assays with a Mass Spectrometric Readout |
title_fullStr | Nanopatterned Extracellular Matrices Enable Cell-Based
Assays with a Mass Spectrometric Readout |
title_full_unstemmed | Nanopatterned Extracellular Matrices Enable Cell-Based
Assays with a Mass Spectrometric Readout |
title_short | Nanopatterned Extracellular Matrices Enable Cell-Based
Assays with a Mass Spectrometric Readout |
title_sort | nanopatterned extracellular matrices enable cell-based
assays with a mass spectrometric readout |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501326/ https://www.ncbi.nlm.nih.gov/pubmed/28120616 http://dx.doi.org/10.1021/acs.nanolett.6b04176 |
work_keys_str_mv | AT cabezasmariad nanopatternedextracellularmatricesenablecellbasedassayswithamassspectrometricreadout AT mirkinchada nanopatternedextracellularmatricesenablecellbasedassayswithamassspectrometricreadout AT mrksichmilan nanopatternedextracellularmatricesenablecellbasedassayswithamassspectrometricreadout |